Flow cytometry

ABSTRACT

A flow cytometer has an aperture in which an inlet chamber and/or an outlet chamber have predetermined geometric relationships with respect to the aperture for decreasing electronic edge effects and increasing the sensitivity of electronic particle volume measurements. In one embodiment, the aperture is triangular and is formed by assembling a plurality of truncated pyramids with their truncated surfaces defining aperture walls. At least one of the elements forming the aperture walls is either part of a lens systems or a transparent cover plate so that simultaneous optical and electronic cell volume measurements can be performed. In one embodiment, a piezoelectric transducer is provided to sonically clear any aperture clogging.

This is a continuation of application Ser. No. 648,356, filed Sept. 7,1984 now U.S. Pat. No. 4,673,288 which was a continuation of applicationSer. No. 263,882 filed May 15, 1981, now abandoned.

BACKGROUND OF THE INVENTION

This invention pertains to flow cytometry, and more particularlypertains to a flow cytometer in which simultaneous optical measurementsand electronic particle volume measurement can be made.

The history of flow particle analyzers is divided into two main lines ofdevelopment, these being optical on the one hand and electronic on theother hand. Further, there have recently been various proposals forcombining the optical and electronic techniques. A basic text coveringthe history of the entire field is FLOW CYTOMETRY AND SORTING: Melamed,Mullaney, Mendelson, et al, John Wiley & Sons; New York; 1979. In orderto facilitate understanding the features and advantages of the presentinvention and by way of a background to it, brief summaries of thedevelopment of the optical, electronic and combined techniques are setforth hereafter.

Optical Flow Particle Analyzers

Optical flow cytometry has been in development over the last 25 years.These systems initially were used to count cells or particles inaccordance with the change in absorbence of an illuminating light as thecells were passed through a glass capillary tube in the measurement pathof a photoelectric measuring device. Later improvements included theprovision of laminar sheath flow of the particles or cells past theobserving area. In this, a suspension of the cells or particles underinvestigation is injected into a faster flowing stream of fluid. Thisfaster flowing fluid provided a sheath around the particles, producinglaminar flow. The laminar flow addition was important because it alloweduse of a large diameter flow stream, thus minimizing clogging andprecisely centering sample streams of particles or cells in an analysisvolume.

The literature concerning developments related to laminar flow andhydrodynamic focusing techniques is discussed in the FLOW CYTOMETERY ANDSORTING text. Patents related to various aspects of this subject includeU.S. Pat. Nos. 3,299,354; 3,661,460; 3,738,759; 3,740,665; 3,810,010;3,871,770; 3,984,307; 4,140,966; 4,162,282; and 4,237,416;

Besides absorption optical measures, both light scatter and fluorescenceare techniques that have also been used to analyze cells or particles.In this connection, see the extensive discussion of these techniques andthe bibliographies contained in the FLOW CYTOMETRY AND SORTING textdiscussed above. Patents related to light scatter techniques includeU.S. Pat. Nos. 3,614,231; 3,646,352; 3,669,542; 3,705,771; 3,785,735;3,786,261; 3,873,204; 3,960,449; 4,053,229; 4,173,415; and 4,188,121.Patents related to fluorescence techniques include U.S. Pat. Nos.3,788,744; 4,225,229; and 4,243,318.

In 1965, Kamentsky et al were the first group to perform two parameterlight measurements on cells by flow techniques. See Kamentsky et al;Spectrophotometer: New instrument for ultrarapid cell analysis, Science150:630-631, 1965. In this work ultraviolet absorption by nucleic acidswas measured while simultaneously analyzing light scatter. There arealso many prior patents directed to multiple optical measurement, seeU.S. Pat. Nos. 3,662,176; 3,850,525; 4,038,556; 3,824,402; 3,916,197.

The sensitivity of optical measurements is related to how much of thefluorescent light emitted by the particle can be collected, or how muchof the incident light which is scattered or absorbed can be recollected.These parameters are related to the optical configuration of theinstrument, including the numerical aperture (N.A.) of the lens systemand whether the optical axis is parallel to or transverse to the streamof flow of the particles or cells.

Optical systems have been described for measuring fluorescence in aparticle flow stream parallel to the optical axis (axial flow). Somesuch systems have advantageously used incident light illumination, wherethe same optics are used to introduce light to the particle flow and tocollect the fluorescent light from the particles.

There have also been described optical transverse flow cytometers, wherethe flow of particles or cells is transverse to the optical axis. See,for example, U.S. Pat. Nos. 4,056,324; 4,225,229; 3,720,470; andSCIENCE, Vol. 204, 27 April 1979, page 403.

Axial Flow optical cytometers, although they have shown some very goodresults with fluorescence measurements, have several disadvantages. Alight scatter parameter is not possible due to the difficulty in placingthe detector behind the cells being measured. Secondly, an objective isnecessary which has a significant depth of focus in order to focusthrough the cross stream trough onto the emerging cells or particles.This arrangement, because of the large depth of field, increases thevertical cell analysis volume significantly, thus limiting the rate atwhich cells can be analyzed. With the increased vertical cell analysisvolume, two or more cells can be introduced into the analysis volume anddetected as a single fluorescent pulse (coincidence problems). Finally,the axial flow design does not lend itself to cell sorting, becausecells exiting the orifice are not caught in a laminar flow stream.

Transverse flow optical cytometers circumvent some of the problemsassociated with axial flow. First of all, light scatter is possible,since detectors can be placed around the incident light beam (i.e. froma laser) which intersects the transverse flow. With this arrangement,light scatter intensities can be measured orthogonally to the incidentbeam. However, these orthogonal systems measure fluorescence 90° to theincident light. The amount of fluorescence measured from a cell at thisangle is minimized due to internal absorbance of the fluorescent lightbefore it exits at right angles to the incident beam. This "darkfield"illumination is used because one is assured of the powerful incidentlaser light not interfering with the fluorescent signal measured at 90°to the incident light. Since these geometrics are used on water streamsin air, the orthogonal flow optical cytometer must use dry objectives,which limit the numerical apertures to 0.7 or less.

ELECTRONIC PARTICLE VOLUME

The basic concept of electronic counting of particles suspended in aconducting fluid passing through a small aperture is disclosed in U.S.Pat. No. 2,656,508 to Coulter. In this, an electric field is appliedacross the aperture or orifice, so as to cause current flowtherethrough. This aperture current is disrupted when a particle passesthrough the aperture, producing a measurable pulse which is used tocount the particle.

In 1958, Kubitschek in Electronic Counting and Sizing of Bacteria,Nature 182:234-235, 1958, attempted to relate the amplitude or area ofthe pulse to the size of the particle. A large number of publicationsfollowed which attempted to explain and characterize this relationship.In a 1969 article, Grover et al (Electrical Sizing of Particles inSuspension, Biophys J., 9:13981414, 1969) described the "edge effects"caused by the compression of the electric field as it passes from alarge volume through a small aperture. That article also described thehydrodynamic flow considerations and the relationship between theparticle volume and the change in the current observed as the particletraversed the aperture.

Particle trajectory sensitivity has been a problem in electronic cellvolume analysis. Attempted solutions to these problems have includedhydrodynamic focusing of the particle stream by directing it along theaxis of the aperture, making the aperture long with respect to itsdiameter, and electronically rejecting any pulse shapes which were notGaussian. Hydrodynamic focusing has been a reasonably effective solutionto trajectory sensitivity, although the requirement for precisecentering of the particle injector with relationship to the aperture hasresulted in transducer designs which are overly complex.

Other difficulties encountered in the design of electronic particledetection transducers include (1) bubbles from the sample, electrodes,or deaeration of the suspending fluid, (2) electrode products and theireffect on biological cells by changes in cell pH and tonicity, (3)so-called "back cursor" pulses caused by particles which have alreadytraversed the aperture circulating back within the region of theaperture outlet and (4) clogging of the aperture. The solution to thebubble and electrode problems have centered around isolating theelectrodes in separate chambers. Electrode products are minimized bylowering the aperture current and providing bubble traps. Back-cursorpulses have been dealt with by electronic discrimination of the longerback cursor pulses, using a second orifice to prevent recirculation, orflushing the outlet chamber with particle fluid.

Aperture clogging has presented a significant problem to these devices.The apertures are usually 50 to 100 microns in diameter and clog easily.Back flushing, fluid and sample filtration, and even a miniaturewindshield wiper mechanism as described in U.S. Pat. No. 3,259,891 haveall been tried, with for the most part unsatisfactory results.

The literature on electronic cell volume instruments includes variousapproaches using other than direct current through the aperture in orderto try to obtain further information on the particles. These include amulti-frequency alternating current device, an alternating currentdevice with electrodes inside the aperture, and combinations ofalternating current and direct current to determine both changes inresistance and capacitance as a particle traverses the aperture.

COMBINED OPTICAL AND ELECTRONIC PARTICLE VOLUME INSTRUMENTS

It should be apparent that more useful information as to thecharacteristics of a particle or cell can be obtained by the use of bothelectronic and optical techniques, than by the use of either separately.In 1970, Leif, in A Proposal for an Automated Multiparameter Analyzerfor Cells, Automated Cell Identification and Sorting, Academic Press,New York, 1970, pp 131-159, proposed an instrument which would performmultiple optical and electronic measurements. Subsequent literaturedescribes several versions of such a device known as AMAC (AutomatedMultiparameter Analyzer for Cells). One version, known as AMAC I, isdescribed by Leif and Thomas in Electronic Cell-Volume Analysis by Useof AMAC I Transducer, Clin. Chem. 19:853-870, 1973. That instrument wasan axial flow, transmitted illumination device, suitable for lightscatter and electronic measurements. While electronic measurements wereachieved with this device, no optical measurements were ever performed.

Steinkamp et al, in A New Multiparameter Separator for MicroscopicParticles and Biological Cells, Rev. Sci. Instr. 44:1301-1310, 1973,described a transducer for performing combined electronic andfluorescence measurements. However, the measurements were not madesimultaneously. The electronic measurement was made first, thendownstream 135 microseconds later the fluorescent measurement was madeorthogonal to the cell flow.

Kachel U.S. Pat. No. 4,198,160 also describes a transducer for bothelectronic cell volume and fluorescent measurements. Kachel employedincident light illumination for fluorescence and used a hydrodynamicfocusing injector to position the particles. It is possible with thisconfiguration to measure both parameters closer to each other, but stillnot truly simultaneously.

The literature includes a description of an AMAC IV transducer, which isequivalent to the Steinkamp apparatus described above, with theinclusion of a water immersion objective to pick up the fluorescenceorthogonal to the laser excitation of the particle stream. The waterimmersion objective has a larger numerical aperture than air objectives(1.0 vs. 0.7 or less) and hence increased the light gathering ability inthe fluorescence measurement. Independent electronic and fluorescencemeasurements were made but no combined measurements were successfulbecause of the time delay from the electronic to the opticalmeasurement.

The prior art of combined electronic and optical measurements alsoincludes U.S. Pat. Nos. 3,710,933, 3,675,768, and 3,770,349. U.S. Pat.No. 3,710,933 to Fulwyler et al describes a system in which theelectronic and optical measurements are made sequentially, and not trulysimultaneously. The U.S. Pat. Nos. 3,675,768 and 3,770,349 to Sanchezdescribe apparatus which is said to provide both electronic measurementsand light absorption measurements.

All of the combined techniques discussed above have used circllarapertures in a wall as the transducer. There is one report in theliterature, however, of an experimental transducer utilizing a squareaperture, although the method by which the square aperture was formedhas not been published. This was the AMAC III transducer, described inR. A. Thomas et al, Combined Optical and Electronic Analysis of CellsWith the AMAC Transducers, Journal of Histochemistry and Cyoochemistry,Vol. 25, No. 7, pp. 827-835, 1977. That transducer used hydrodynamicfocusing to direct the particle stream and orthogonal fluorescentdetection to the laser excited stream. The light measurements were madein a miniature cuvette which was the electronic aperture, so thattheoretically all measurements could be made simultaneously. Separateelectronic and fluorescent measurements have been reported, but to dateno report of simultaneous measurements have been made.

Several common problems have existed in prior art attempts at combinedelectronic and optical cytometry. Unless the measurements are truly madesimultaneously, there is a coincidence problem involved in accuratelyrelated the two sequential measurements to the same particle. If it isattempted to truly make simultaneous electronic and opticalmeasurements, conflicting design considerations are involved.

With the electronic measurement, the aperture resistance must be largecompared to the inlet and outlet resistance. If this is not the case,the effective length (i.e. the distance during which the particleappreciably affects the current flow) becomes long and the coincidencerate becomes unacceptable. The particles must pass rapidly from a regionof low current flux to a region of high current flux and back again to aregion of low current flux. In addition, they must travel along atrajectory which avoids the "edge effects."

With the optical measurements the larger the numerical aperture (N.A.)the more sensitive is the measurement. That is, resolving power isdirectly proportional to N.A., image brightness is proportional to(N.A.)², and depth of focus is inversely proportional to N.A. However, alarge N.A. implies a large acceptance angle, which dictates that theobject being measured must be as close as possible to the collectinglens.

In any axial flow combined transducer configuration, the electronic andoptical design configurations are at odds with each other, andconcessions must be made which reduces the sensitivity of either or bothmeasurements. For example, in order to maximize the outlet volume forelectronic considerations, the objective must be moved away from theaperture, which results in either a decrease in N.A. or loss ofsimultaneity of measurement, or both. In the transverse flow geometry,downstream optics has been the only solution, which results in the lossof simultaneity of the measurements.

Of all of the prior art constructions, the AMAC III transducer cameclosest to solving the problems of combined electronic and opticalmeasurements. In the AMAC III the square electronic aperture was alsothe optical aperture. By using a square geometry, the inlet and outletchambers were large in volume with respect to the aperture volume, thusaccommodating the electronic constraints. The square geometry, however,sacrificed N.A. by limiting the acceptance angle to 90° for thefluorescent measurement. The fluorescence was activated by anorthogonally disposed laser which sacrificed the light gained byincident light illumination, but produced good light scattercharacteristics.

As can be seen from the foregoing abbreviated discussion of some of theprior art, the field of flow cytometry has and continues to be veryactive. An unquestioned need exists for a flow cytometer which canprovide truly simultaneous electronic and optical measurements, withoutsacrificing any of the sensitivity possible with either technique.Additionally, the need exists for improved sensitivity in bothelectronic and optical measurement techniques.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a method andapparatus for improved sensitivity flow cytometry.

It is another object of this invention to provide a method and apparatusfor flow cytometry which simultaneously provides electronic and opticalmeasurements.

It is another object of this invention to provide a flow cytometertransducer in which inlet and outlet geometries are such as to decreaseelectronic edge effects through the aperture while providing increasedelectronic sensitivity.

It is another object of this invention to provide an electronic andoptical flow cytometer transducer formed of a plurality of solidpolygons defining a polygonal aperture.

It is another object of this invention to provide a flow cytometer forsimultaneous electronic and light scatter measurements.

It is another object of this invention to provide a flow cytometer forsimultaneous electronic and optical measurements in which at least oneof a plurality of elements defining the cytometer aperture is the firstelement in the optical system for making the optical measurements.

It is another specific object of one embodiment of this invention toprovide a flow cytometer having a triangular transducer aperture.

It is another object of this invention to provide a flow cytometer andmethod in which sonication is used as a means for clearing a cloggedaperture.

It is another object of this invention to provide a flow cytometer forsimultaneous electronic and optical measurements in which incident lightillumination and sensing is used for the optical measurements.

Briefly, in accordance with one embodiment of the invention, there isprovided a flow cytometer having an aperture in which an inlet chamberand an outlet chamber are provided which have predetermined geometricrelationships with respect to the aperture for decreasing electronicedge effects and increasing the sensitivity of electronic particlevolume measurements. In accordance with one of the embodiments, theaperture is triangular and is formed by assembling a plurality oftruncated pyramids with their truncated surfaces defining walls of theaperture. The inlet and outlet geometries formed by the truncatedpyramids are configured to reduce edge effects from the flow of currentthrough the aperture and to maintain a predetermined cross-sectionalarea relative to the cross-sectional area of the aperture. Electrodesare provided to establish current flow through the aperture, forelectronic particle or cell volume measurements. At least one of theelements forming the aperture walls is either part of a lens system or atransparent cover plate for an oil immersion objective used inperforming simultaneous optical measurements on particles or cells whilethey are in the aperture. These optical measurements can befluorescence, light scatter, or both. Further, in accordance with oneaspect of one embodiment of the invention, a piezoelectric transducer isprovided to sonically clear any clogging of the aperture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-section of a flow cytometer aperture in accordancewith the prior art, illustrating the current flux density edge effects.

FIG. 2 is a schematic representation of a flow cytometer aperture havingan inlet chamber with angled walls.

FIG. 3 is similar to FIG. 2 and illustrates various angularrelationships concerning a flow cytometer aperture.

FIG. 4 is a graph illustrating edge effect current flux variations asthe angle of the inlet chamber wall varies.

FIG. 5 is a graph of the ratio of the inlet chamber cross-section to theaperture cross-section for various distances into the inlet chamber, forvarious configurations of inlet chambers.

FIG. 6 is a front elevation of one embodiment of a flow cytometeraccording to the invention.

FIG. 7 is a side elevation of the cytometer of FIG. 6.

FIG. 8 is a cross-section of a portion of the cytometer of FIG. 6.

FIG. 9 is a cross-section along the line 9--9 of FIG. 8.

FIG. 10 is a top plane view of truncated pyramids forming elements inthe cytometer of FIG. 6.

FIG. 11 is a front elevation showing the truncated pyramids of FIG. 10joined together.

FIG. 12 is a bottom view of the assembly of FIG. 11.

FIG. 13 is partly a cross-section of a portion of the cytometer of FIG.6 and illustrates diagrammatically an optical sensing arrangement aspart of the cytometer.

FIG. 14 is a diagrammatic representation of a flow cytometer opticalsensing arrangement in accordance with one embodiment of the invention.

FIG. 15 is similar to FIG. 14 and illustrates another embodiment of anoptical sensing arrangement.

FIG. 16 is a front elevation partly in section of a transducer for anelectronic flow cytometer.

FIG. 17 is a section of a portion of the transducer of FIG. 16.

FIG. 18 is a view along the axis of the inlet chamber of the transducerof FIG. 16.

FIG. 19 is a view showing the triangular inlet chamber and aperture ofthe transducer of FIG. 16.

FIG. 20 is a block diagram of suitable electronics for use with thecytometer of the present invention.

FIG. 21 is a combined electronic particle-fluorescence histogram of DukeScientific 10 micron diameter fluorescent spheres.

FIG. 22 is a flow cytometric DNA histogram of nuclei from Wistar ratliver.

FIG. 23 is a flow cytometric DNA histogram of nuclei from normal CF1mouse kidney.

FIG. 24 is a flow cytometric DNA histogram of nuclei from normal CF1mouse liver.

FIG. 25 is a flow cytometric DNA histogram of nuclei from normal humanbreast tissue.

FIG. 26 is a flow cytometric DNA histogram of nuclei from a patient withbreast cancer.

FIG. 27 is an electronic cell volume plot of Wistar rat liverdissociated with enzymes.

FIG. 28 is a combined plot of electronic nuclear volume and DNAfluorescence of nuclei from a patient with breast cancer.

FIG. 29 is a combined plot of electronic nuclear volume and DNAfluorescence of nuclei from Wistar rat liver.

FIGS. 30(a) and 30(b) are electronic nuclear volume and intensity offluorescence histograms for CF1 mouse liver nuclei.

FIG. 31 is a combined plot of electronic nuclear volume and DNAfluorescence of CF1 mouse liver nuclei.

FIGS. 32 and 33 are reproductions of actual oscilloscope tracings forsimultaneous electronic nuclear volume and fluorescence pulses for mouseliver nuclei.

DETAILED DESCRIPTION

In its broadest aspect, the present invention contemplates providing anaperture or sensing zone for electronic or combined electronic/opticalflow cytometers in which an inlet chamber and an outlet chamber areprovided adjacent the aperture and which have predetermined geometricrelationships to the aperture. The predetermined geometries of the inletand outlet chambers in accordance with the invention decrease theelectronic "edge effects" and also result in significantly increasedsensitivity of electronic volume measurements of sample particlespassing through the aperture.

The prior art of electronic particle volume counters has provided anaperture or sensing zone in the form of a small circular hole providedin a wall. FIG. 1 illustrates diagrammatically the pattern of thecurrent flux density through such an aperture consisting of a circularhole through a wall 1. As can be seen, the current flux density has atendency to bunch up at the edge of the circular hole. these "edgeeffects" are responsible for a large amount of false information whenattempting to relate the magnitude of the change in current to the sizeor volume of the particle. For example, slight trajectory differences asparticles traverse the measuring zone result in different amounts ofcurrent being blocked by identically sized particles.

In accordance with the present invention, an inlet and an outlet chamberare provided to the aperture which have chamber walls providedimmediately adjacent the aperture that have a predetermined geometricrelationship to the aperture.

In accordance with a first aspect as shown in FIGS. 2 & 3 insofar asdecreasing the edge effects, assume a chamber 2 where the current fluxdensity is uniform. This chamber connects to an inlet chamber havingwalls 3 making an angle θ with respect to the plane of an aperture ormeasuring zone 4. Since current follows the line of least resistance, itis possible as a first order approximation to stipulate that the currentflux passing through area Z₁ of chamber 2 will pass through the volumesubtended by the angle Φ₁ the current flux passing through Z₂ will passthrough the volume subtended by Φ₂, and correspondingly for Z_(n) andΦ_(n). Hence, the number of degrees represented by Φ_(n) will beinversely proportional to the current flux passing through Z_(n). Forfixed values of I_(n) (representing a percentage of the width of themeasuring zone or aperture inlet), it is possible to calculate thenumber of degrees for each corresponding angle Φ_(n) for varying valuesof the angle θ. For the prior art arrangement of the hole in the wall, θequals zero. For θ equals to 90°, a measuring chamber with no inletangle would be described.

Referring to FIG. 3, assume for the sake of discussion, that theaperture is 100 microns wide, and that an angle φ₅₀ is defined as theangle formed from the center of the aperture to its edge (with the inletchamber wall at an angle θ). Additional angles φ₄₀, φ₃₀, φ₂₀, φ₁₀ arethe angles formed between the center of the aperture and 40, 30, 20, 10microns from the center, respectively. The distance L shown in FIG. 3can be described in terms of θ by ##EQU1## Therefore, the L can bedetermined by varying θ. As the angle θ increases or decreases, the Lvalues can be determined. Therefore, the flux line density can bedescribed at the aperture entrance at arbitrary distances along thewidth of the aperture from the center by determining φ for a given θ (orL which is used in the calculation). These arbitrary distances can, forexample, be multiples of I, which can be 10 μm.

The value for φ_(n) is then given by ##EQU2##

Table I given below shows the result of this approach. The entrance ofthe measuring zone or aperture has been broken into ten equal widthsegments, five to either side of the center of the aperture. Thequantity 1/φ₁₀ represents a segment from the center moving toward theedge 10% of the width of the aperture; 1/φ₂₀ is from 10%-20% of thewidth; 1/φ₃₀ is from 20%-30%; 1/φ₄₀ is from 30-40% of the width; and1/φ₅₀ is from 40-50%, i.e., the edge of the aperture.

                  TABLE I                                                         ______________________________________                                        Distance into the orifice (L) vs. the inverse of φ at various             distances from the centerpoint at the beginning of the orifice                At 10 μm from the center (I = 10)μ.sub.m to the edge (I                 = 50μ.sub.m)                                                               φ                                                                              L        1/φ.sub.10                                                                         1/φ.sub.20                                                                       1/φ.sub.30                                                                      1/φ.sub.40                                                                      1/φ.sub.50                      ______________________________________                                         1°                                                                         0.873    0.0118   0.402  1.205 2.381 4.000                                2°                                                                         1.746    0.0125   0.204  0.602 1.205 2.000                                3°                                                                         2.620    0.0133   0.139  0.405 0.806 1.333                                4°                                                                         3.496    0.0141   0.107  0.306 0.602 1.000                               10°                                                                         8.816    0.0206   0.057  0.135 0.253 0.412                               20°                                                                         18.199   0.0347   0.053  0.090 0.148 0.224                               30°                                                                         28.870   0.0523   0.064  0.088 0.124 0.172                               40°                                                                         41.955   0.0746   0.083  0.099 0.124 0.157                               50°                                                                         59.588   0.105    0.111  0.122 0.140 0.163                               70°                                                                         137.37   0.240    0.243  0.248 0.256 0.265                               85°                                                                         571.50   1.00     1.00   1.00  1.00  1.00                                87°                                                                         954.10   1.67     1.67   1.67  1.67  1.67                                89°                                                                         2864.5   5.00     5.00   5.00  5.00  5.00                                ______________________________________                                    

As can be seen from Table I, for an inlet angle of 1° (essentially ahole in a wall) the variation in values 1/φ₁₀ to 1/φ₅₀ is quite large,with very large values near the edge, i.e., for 1/φ₅₀. As the inletangle θ is increased, the dispersion (difference across the measuringinlet in current flux density) decreases. At a θ of 50°, the values arealmost constant across ##EQU3## FIG. 4 is a plot of these values. As canbe seen from FIG. 4, the curves converge at around 50° and remainconverged to 90°. As can be seen from the curves of FIG. 4, significantreduction of the edge effects result from inlet chamber angles θ of 5°and greater. Thus, in accordance with this aspect of the invention, aninlet chamber is provided to the sensing chamber with the inlet chamberwalls disposed at an angle of 5° or greater with respect to the plane ofthe aperture, for reducing edge effects. Like considerations apply toproviding an exit chamber whose walls are disposed at an angle of 5° orgreater with respect to the plane of the aperture.

In addition to decreasing edge effects in electronic particle volumecytometers, it is desirable to have as sensitive an instrument aspossible. The current flux density in the aperture is inverselyproportional to the cross-sectional area. Hence, when the particle isconfined to a small geometrical cross-section, the current fluxinterrupted by the particle will be maximized. Therefore, as theparticle is travelling toward the measuring zone (i.e., the place wherethe current flux density is maximized), it is desirable for it to passrapidly from a region of low current flux density to a region of highcurrent flux density, i.e., to rapidly pass from a zone where it makesno appreciable change in the current to a zone where the change incurrent is a maximum.

In accordance with the principles of this invention, inlet and outletchambers are provided adjacent either side of the sensing aperture, withthe cross-sectional area of the inlet and outlet chambers having apredetermined relationship to the cross-sectional area of the aperture.First, a size parameter, which will be called an "S" unit, is defined asthe maximum linear dimension or width of the aperture along any planepassing through the axis of the aperture. In accordance with this aspectof the invention, the aperture can have any cross-sectional geometricconfiguration, i.e., circular, square, triangular, etc. It has beenexperimentally determined that at a linear distance of two "S" unitsmeasured from either side of the aperture into the inlet and outletchambers, that the cross-sectional area of the inlet and outlet chambersat these points should be greater than ten times the cross-sectionalarea of the aperature. (See FIG. 5) If the cross-sectional areas of theinlet and outlet chambers at these points is greater than ten times thecross-sectional area of the aperture itself (and preferably on the orderof twenty-thirty times as much in cross-sectional area), it has beenfound that sharp, well-defined, electronic particle volume pulses fromthe electronic cytometer result.

In accordance with another aspect of this invention, an improved methodhas been found for constructing electronic and/or optical sensingapertures for flow cytometers by cooperative relationship of a pluralityof solid polygons. The terms "solid polygons" is meant to refer to amulti-sided three-dimensional polygonal shape. A plurality of thesesolid polygons are provided having surfaces which function to define theaperture or sensing zone, and also having surfaces defining the geometryof the inlet chamber and the outlet chamber so that they havepredetermined geometric relationships with respect to the aperture orsensing zone, in accordance with the broader aspects of the invention.It is within the scope of the invention to provide such an assembly inwhich the aperture is triangular, square, 5-sided, etc. A triangularaperture is, however, the preferred embodiment for the many advantagesit provides. For making optical measurements, at least one of theelements defining the walls of the aperture is part of an opticalsystem. In accordance with the invention, the at least one element whichis part of an optical system can be a transparent cover plate againstwhich, i.e. an oil immersion objective is situated, or can itself be alens forming the first optical element in an optical system. If desired,it is within the scope of the invention to provide more than one of theelements defining the walls of the aperture, even all of them, as a partor parts of an optical system for either introducing exciting light intothe sensing zone in the aperture or collecting light therefrom.

FIG. 6 shows a front elevation of a portion of an actual apparatusconstructed in accordance with principles of the present invention. FIG.7 is a side elevation thereof, and FIGS. 8, 9 and 13 are varioussectional views thereof. In the drawings, a mounting plate 11 mounts viaa clamp member 12 two electrolyte reservoirs 13 and 14. The electrolytereservoir 13 is in communication with a suitable source of electrolytethrough conduit 16. The electrolyte reservoir 14 is coupled through itsconduit 17 to a repository for used electrolyte, which may include avacuum source or the like for establishing a negative pressure.Altlernatively, a pressure can be coupled to the inlet reservoir toestablish the required pressure differential between the inlet andoutlet reservoirs. Disposed within the reservoir 13 (which is theelectrolyte supply side of the device) is an electrode 18. The electrode18 can be foil electrode or the like and is electrically connected to alead 19. In a similar fashion, the reservoir 14 has mounted therein anelectrode 21 which is electrically connected to a lead 22.

Attached to the bottom of reservoir 13 is a housing 23. The housing 23has a passageway generally indicated by reference numeral 24 formedtherein, which is in communication with the interior of reservoir 13.Similarly, a housing 26 is provided mounted to the bottom of reservoir14 and having a passageway generally indicated by reference 27 numeraltherein, which is in communication with the interior of reservoir 14.The housings 23 and 26 have O-ring assemblies 28 and 29, respectively.

The O-ring assemblies 28 and 29 plug into and mount ceramic blocks 31and 32. A glass solid polygon 33 is adhesively secured to surfaces 31aand 32a of the ceramic blocks 31 and 32. As used herein, the term "solidpolygon" is meant to refer to a multisided three dimensional geometricshape. In a similar fashion, another solid polygon 34 formed of glass isadhesively secured to opposite surfaces of the ceramic blocks 31 and 32(see sectional view in FIG. 7.)

In a manner more fully discussed hereafter, the glass solid polygons 33and 34 cooperate to define an aperture at a sensing zone generallyindicated by reference numeral 36 in FIG. 6. Further, in a manner morefully discussed hereafter, the solid glass polygons 33 and 34 areconfigured so as to provide inlet and outlet chambers to the aperturewhich have a predefined geometric relationship to the dimensions of theaperture.

The ceramic block 32 is provided with an inlet passageway 37 whichcouples the passageway 24 to the inlet chamber defined by the glasssolid polygons 33 and 34. In a similar fashion an outlet passageway 38extends through the ceramic block 31 to couple an outlet chamber definedby the glass polygons 33 and 34 to the passageway 27.

In the specific arrangement shown in and described, two flat surfaces ofthe solid glass polygons 33 and 34 define two walls of a triangularaperture at the sensing zone 36. If desired, an identical third solidglass polygon could be provided to form the third wall of the triangularaperture and the third wall of inlet and outlet chambers. In accordancewith the specific embodiment illustrated in the drawings, however, adifferently configured solid glass polygon in the form of a glass coverplate 39 is provided adhesively secured to the solid glass polygons 33and 34 and the ceramic blocks 31 and 32 and functioning to define thethird wall of the triangular aperture at the sensing zone 36 and thethird wall of the inlet and outlet chambers. In this specificembodiment, a coupling 41 is illustrated which is adapted to couple amicroscope objective, such as an oil immersion objective, for opticalviewing of the sensing zone 36 through the glass cover plate 39.

As shown in the drawings, the ceramic block 32 has an additional sampleinlet passageway 42 which mounts through a suitable O-ring assembly 43 asample injector 44. The sample injector 44 extends into the passageway37 adjacent the beginning of the inlet chamber defined by the solidglass polygons 33 and 34. The sample injector 44 is adapted to becoupled to a sample source of particles or biological cells or the like,in a manner well known to those skilled in the flow cytometry art. Itshould be noted as shown in the drawings, that the sample injectorextends at an angle with respect to the flow path of electrolyte throughpassageway 37 and the inlet chamber defined by solid glass polygons 33and 34. Suitable provision is made via, for example, the O-ring assembly43, for longitudinally adjusting the sample injector 44 so as toposition it at various points in the stream of electrolyte flowingthrough passageway 37 and the inlet chamber defined by the solid glasspolygons 33 and 34.

In accordance with a particular aspect of one embodiment of theinvention, a sonicator in the form of a piezoelectric crystal 46 isprovided attached to the mounting plate 11 and having suitable leads 47for connection to a source of electrical power. Also as part of thesonication apparatus, a spring assembly 48 can be provided.

The manner in which the solid glass polygons 33 and 34 cooperate todefine the polygon shaped aperture and the inlet and outlet chambers canbest be understood by referring to FIGS. 10, 11 and 12. FIG. 10 showsthe solid glass polygons 33 and 34 which, in accordance with thisspecific embodiment, are truncated pyramids. As illustrated, the solidglass polygon 33 has four sides 33a, 33b, 33c, and 33d. In a similarfashion, the solid glass polygon 34 (which ca be identical to 33) hasfour sides 34a, 34b, 34c and 34d. Each of the solid glass polygons 33and 34 have truncated portions generally indicated by 33e and 34e. Asshown in FIG. 11, the two solid glass polygons 33 and 34 can be joinedtogether by adhesively securing the surfaces 33d and 34d to one another,with the solid glass polygons in aligned relationship. When this isdone, the truncated portions 33e and 34e define (as shown in thisparticular example) two walls or sides of a triangular shaped sensingaperture. To the arrangement of FIG. 11, and referring to FIG. 6, theglass cover plate 39 is then attached to the bottom of the assemblyshown in FIG. 11 to define the third wall o side of the triangularshaped sensing aperture. It should be clear from FIGS. 10, 11 and 12,that the truncated portions 33e and 34e define both the cross sectionalarea of the sensing aperture and its depth, so that they fix the volumeof the sensing aperture.

As apparent from the drawings, adjacent surfaces 33c and 34a define thevolume and cross-sectional area of either an inlet chamber or an outletchamber, and the corresponding adjacent surfaces 33a and 34c define theother of the inlet or outlet chambers.

Referring back to FIGS. 6-9, the manner in which the illustrated devicegenerally functions is as follows. A flow of electrolyte is establishedthrough the sensing zone 36 (a triangular aperture in this specificembodiment) from the electrolyte reservoir 13 and back up intoelectrolyte reservoir 14. This flow of electrolyte is established bymeans such as a vacuum source connected to the conduit 17 of reservoir14. A source of electric potential A.C. and/or D.C. is connected betweenleads 19 and 22 so that a current flow is established through theelectrolyte between electrodes 18 and 21 via the various passageways andthe sensing zone or aperture 36. Samples, such as particles orbiological cells are injected into the flow of electrolyte through thesample injector 44. As the samples are injected into the flow ofelectrolyte going through the aperture or sensing zone 36, upon passagethrough the aperture they affect the electrical resistance betweenelectrodes 18 and 21 in a manner well known to those versed in the artof flow cytometry generating pulses indicative of particle volumemeasurements.

In accordance with one aspect of the invention, as more fully discussedhereafter in connection with FIG. 13, simultaneous optical measurementscan be made on a particle or biological cell as it is traversing throughthe aperture or sensing zone 36. That is, the glass cover plate 39itself forms one of the walls of the aperture and is part of an integraloptical system focused o the particle or cell while it is in theaperture. In this manner, truly simultaneous electronic and opticalmeasurements can be made on the same exact particle, and no correlationproblems arise.

The angles and dimensions of the various surfaces on the solid glasspolygons 33 and 34 influence both the fluidic flow characteristics andthe electrical field characteristics through the sensing zone oraperture 36. As known in the art, it is important to have laminar flowof the electrolyte containing the samples through the sensing zone oraperture in order to minimize turbulence which would disrupt theposition of the particles in the sensing zone. It has been found thatthe triangulated laminar flow sheath provided by the triangular apertureand triangular inlet and outlet zones formed in accordance with oneembodiment of the invention, provides exceptional positional stabilityto an injected stream of particles injected via sample injector 44.

The angle of the walls of the inlet and outlet chambers influence boththe fluidic flow characteristics and the electrical fieldcharacteristics of the device. The inlet and outlet chamber shapes canbe calculated to produce a continuous fluidic boundary layer from inlet,through the sensing zone, and through the outlet chamber, in order tominimize turbulence. In addition, this shape produces a smoothtransition from a region of low current flux (i.e. the inlet chamber) toa region of high current flux (i.e. the sensing zone or aperture) andback to a region of low current flux (i.e. the outlet chamber). Thissmooth transition decreases the "edge effects" which an abrupttransition from large volume to small volume (i.e. the prior artprovision of a hole in a wall) induce. By limiting the edge effects, theparticle will encounter a region of uniform current flux during itstransit through the aperture or sensing zone. This leads to improvedsensitivity of the electronic measurements.

In accordance with the broader aspects of the invention, the walls ofthe inlet and outlet chambers are configured such that they make anangle of 5° or greater with respect to the plane of the aperture inorder to decrease the edge effects. Further in accordance with thebroader aspects of the invention the walls of the inlet and outletchambers are configured such that at a distance of two "S" units awayfrom the sides of the sensing aperture the cross-sectional area of theinlet and outlet chambers is at least ten times greater than thecross-sectional area of the aperture. These geometric considerationsdictate the limit conditions for the geometries of solid polygons usedto define the aperture and the inlet and outlet chambers in accordancewith that aspect of the invention.

In accordance with the embodiments of the invention wherein solidpolygons are used to define the aperture and the inlet and outletchambers, as discussed previously truncated pyramids have been found tobe advantageous geometries for the solid polygons, although it is notintended to be limited thereto. Using truncated pyramids, either atriangular aperture and inlet and outlet chambers can be formed byassembling three identical truncated pyramids, or a square aperture andinlet and outlet chambers can be configured by assembling four truncatedpyramids. FIG. 4 is a plot of the ratio between the cross-sectional areaof the inlet chamber to the cross-sectional area of the aperture versus"S" units away from the entrance of the aperture or sensing zone fortriangular and square orifices assembled by joining a plurality oftruncated pyramids. The angular references on the curves in FIG. 4 referto the angles made by the flat walls of the inlet and outlet chamberswith respect to the axis of the aperture. Using curves like those ofFIG. 4, the angles for the truncated pyramids used to assemble thetransducer can be calculated in accordance with the desired relationshipbetween the inlet and outlet chamber cross-sections and that of theaperture, in accordance with the principles of this invention. As can beseen for the case of the triangular aperture with a 30° inlet angle, attwo "S" units away from the measuring zone, a particle influences thecurrent by about three percent of the maximum value. This is derived bythe ratio of 31/1 of inlet cross-sectional area to measuring zonecrosssectional area. Therefore, a particle in the measuring zone willexperience a three percent coincidence error by a following particlewhich is two "S" units away from the measuring zone.

In the specific embodiment of the invention shown in FIG. 6, the wallsof the two truncated pyramids defining two walls of the inlet and outletchambers were oriented at 50° with respect to the plane of the aperture,leading to negligible edge effects in the electronic measurements.Further, the ratio between the cross-sectional areas of the inlet andoutlet chambers at two "S" units from the aperture sides wasapproximately 40 to 1. This was found to result in sharp, well definedelectronic particle volume pulses.

It should be apparent from the drawings, that in accordance withspecific embodiments of the invention the flow of electrolyte containingsamples from the inlet chamber through the aperture or sensing zone andout the outlet chamber is an arch-shaped flow, with the aperture orsensing zone at the vertex or "knee" of the arch. This is important fromthe standpoint of optical sensing. As clear from the drawings, opticalsensing is through the glass plate 39 forming one of the walls of thesensing zone. Since the particle path is arched, the particles in thesensing zone remain in focus as they traverse across the optical paththrough the aperture or sensing zone. Particles which either have notyet reached the sensing zone or which are leaving the sensing zone arenot in focus, and hence do not interact with the optical measurement ofparticles in the sensing zone.

In accordance with one particular feature of one embodiment of theinvention, the sample injector 44 is not coaxially centrally located, ashas been the case in the prior art. Rather, the sample injector isbrought in at an angle to the triangulated flow sheath of theelectrolyte. As the particle injector is moved from edge to edge of thetriangulated flow sheath, the position of the particles in the sheathand hence in the sensing zone or aperture is changed. In this manner,precise positioning of the sample stream in the electrolyte in order tofurther minimize electronic field edge effects experienced by theparticle stream can be accomplished.

The provision of a triangular-shaped sensing zone or aperture, besidesthe advantages it offers by way of imparting exceptional stability tothe triangulated flow sheath, has other advantages. In electronicdetection of particle characteristics, the volume of the sensing zone oraperture (i.e. the cross sectional area times the length) is one of thedetermining factors in the sensitivity of the measurement. For a smallparticle you need a small cross section for greatest sensitivity. Thesmaller the cross section, however, the greater is the tendency to clog.Hence it is advantageous to have a small cross-section yet as large aspossible a dimension across the cross section, in order to decreaseclogging. For the case of an equilateral triangle, the longest dimensionis 1.35 times as great as a circle with the same cross sectional area.In accordance with a specific embodiment of the invention, thetriangular orifice or aperture was such that it was 100 microns on aside and 100 microns long.

The geometry of a triangular aperture also offers significant advantageswith regard to optical measurements on particles in the aperture. Thesensitivity of any optical measurements is related to how the quantityof total light, which is emitted from the particle, can be collected, orhow much of the incident light, which is scattered or absorbed, can berecollected. Using a triangular aperture sensing zone, for the case ofan equilateral triangle, 120° per side of light gathering can beachieved in the axis of the plane of the triangular cross section. Inthe axis perpendicular to that, 180° is available, depending on thegeometry of the inlet hole.

One method of establishing a practical optical boundary is byconsidering the state of the art in available microscope objectives. Todate, high quality objectives have a Numerical Aperture (N.A.) of 1.25to 1.4. The N.A. is given by the formula:

    N.A.=n.sub.o sin θ

where n_(o) is the index of refraction of the object space and θ is thehalf angle of acceptance of light. Since these lens are maximized towork in an objective space of n_(o) =1.515, then a range of θ may bedetermined for these lens, i.e., N.A.=1.25=1.515 sin θ. ##EQU4## so apractical optical geometrical limit for the best commercially availablemicroscope optics is a half angle of light acceptance between 55° and67°. This fits right in the range of acceptance angle provided by oneside of a triangular aperture.

In accordance with one specific embodiment of the invention shown inFIG. 13, in which the optics are indicated in diagrammatic form, oilimmersion objective 51 is provided adjacent the glass cover plate 39,and focused on the sensing zone or aperture 36. A suitable light source52 is provided for introducing exciting light into the sensing zone viameans such as beam splitter 53. Assuming that fluorescent measurementsare to be made with respect to samples in the sensing zone 36, the lightsource 52 can either be a mercury bulb or a laser, emitting light, forexample, in the ultraviolet spectrum. In any event, the exciting lightis introduced through the oil immersion objective 51 into the sensingzone or aperture 36. Assuming that the samples flowing through theaperture or sensing zone 36 have been properly prepared, i.e. stained orthe like in the case of biological cells, they will emit characteristicfluorescent light. This light is collected by the oil immersionobjective 51 and passed through the beam splitter 53 to an opticaldetector 54, such as a photo multiplier tube or the like. Since in thespecific embodiment being discussed the aperture is triangular, light iscollected by the oil immersion objective 51 over an acceptance angle of120°, providing a N.A. of 1.3. As an alternative to providing the glasscover slip 39 as part of the optical system, the element defining thatwall of the aperture or sensing zone can be a lens itself.

In the descriptions of the solid polygons 33 and 34 and cover plate 39,they have been referred to as "glass." No particular limitation isintended by the use of such term, the point being that the elementsdefining the walls of the sensing aperture have optical qualities suchthat they can form part of a system for making optical measurements withrespect to particles in the sensing aperture. In a particularembodiment, the solid polygons 33 and 34 were made from optical flats ofBK7 glass, with precision lapping at desired angles to form the desiredtruncated pyramids. Whatever the material chosen for the solid polygons33 and 34, care should be taken to use a material for the blocks 31 and32 (FIG. 1) that has an equivalent thermal coefficient of expansion.

In the description of FIG. 7, reference was made to a piezoelectriccrystal 40. It has been found that providing such a sonication apparatusis a very effective means of clearing any clogs that might arise in thesensing aperture 36. Temporarily applying electrical power to thepiezoelectric crystal set up sonic vibrations through the electrolyte inthe cytometer, and has been found to be very effective in clearing clogsthat occur at the sensing aperture 36. As an alternative to providing apiezoelectric crystal affixed to the cytometer mounting plate as shownin FIG. 7, a piezoelectric crystal or other sonication producing elementcan be coupled to the sample injector 44. This would serve to set uplocalized sonication in the electrolyte near the sensing aperture 36.

Suitable electronic circuits for processing, recording and displayingdata from the electronic and optical measurements are described in theliterature and known to those skilled in the flow cytometry art. FIG. 20illustrates in block diagram form one exemplary electronic arrangementsuitable for this purpose. The electronic channel can include pulsestorage and shaping circuitry 106, followed by amplifier/filtercircuitry 107, and an analog to digital converter 108 which couple theelectronic measurement signal into a multiparameter analyzer 109. Theoptical channel includes a suitable optical detector 111, coupledthrough amplifier/filter 112 and analog to digital converter 113 to themultiparameter analyzer 109. If desired, a real time display circuit 114can be provided to provide a display of the relatively unprocessedsignals from the electronic and optical channels.

FIG. 20 illustrates only one optical channel feeding into themultiparameter analyzer 109. Of course, if more than one opticaldetector is employed as in various of the embodiments of the invention,a corresponding plurality of optical channels can be provided.

As is conventional, the multiparameter analyzer 109 inputs to a suitablecomputer 116 which can perform various data manipulation andcorrelation. Connected to the computer can be data storage means 117 anda graphics means 118 for providing graphs and the like.

FIG. 14 is a diagrammatic representation of one of the many possiblealternate embodiments of this invention in which more than one side ofthe triangular aperture or sensing zone can be used for making opticalmeasurements. In FIG. 9 the triangular aperture or sensing zone 56 isshown as formed by flat surfaces of three solid polygons 57, 58 and 59.The solid polygons 57, 58 and 59 can be made of glass and have flatsurfaces 57a, 58a, and 59a defining the walls of the triangular apertureor sensing zone 56. The solid polygons themselves can be ground so thatthey function as lenses for the respective sides 57a, 58a, and 59a ofthe aperture. Such additional lens elements as necessary can be providedin the optics for the three sides of the aperture 56, but are not shownin FIG. 14 for simplicity. Referring to the side of the aperture definedby surface 57a, means can be provided for introducing exciting lightinto the aperture to excite a particle (indicated by the dashed line 60)by means of a light source 61 and a beam splitter 62. Means such as anoptical detector 63 are provided for detecting the light emitted byparticle 60 through the wall 57a of the aperture 56.

In a similar fashion, means can be provided in the form of a lightsource 64 and a beam splitter 66 for introducing exciting light into theaperture through the wall 58a thereof. Likewise, an optical detector 67can be provided for detecting the light emitted by the particle 60through the wall 58a of the aperture. In the same fashion, a lightsource 68 and beam splitter 69 can be provided for introducing excitinglight through the wall 59a of the aperture. Means such as an opticaldetector 71 can be provided for detecting the light emitted by theparticle through the wall 59a.

The light sources 61, 64, and 68 can all be identical, i.e. ultra violetsources, for fluorescent measurements or the like on the particle 60, orthey can be different. Further, if desired, only one light source can beprovided, with detectors being provided on two or all three sides of theaperture 56. The point is that by collecting light on all three sides ofthe aperture 56, an effective N.A. of three times the N.A. for one side,i.e., 3.9, can be achieved, vastly improving the sensitivity of opticalmeasurements.

Various different schemes for using only one light source and oneoptical detector, while at the same time increasing the effective N.A.are possible. Thus, in FIG. 14, only the one light source 64 and oneoptical detector 67 could be provided for introducing exciting lightthrough the aperture surface 58a and collecting light from the particle60 through the surface 58a. To increase the effective N.A., the surfaces57a and 59a can be mirror surfaces or the other surfaces of the polygons57 and 59 can be mirrored, so as to reflect all light emitted by theparticle 60 back through the surface 58a of aperture 56 for detection bythe optical detector 67. Alternatively or additionally, the mirroredsurfaces can function to reflect portions of the exciting light onto theparticle 60.

Turning to FIG. 15, there is shown still another possible embodiment ofthe invention in which light scatter measurement techniques are combinedwith fluorescent measurement techniques. In FIG. 15, solid polygons 72and 73 are provided having flat surfaces 72a and 73a defining two wallsof a triangular aperture or sensing zone 74. In the specific embodimentshown in FIG. 15 a glass cover plate 76 is shown as constituting thethird wall 76a of the aperture 74, although it should be understood thatinstead of the glass cover plate 76 another solid polygon could beprovided. With regard to the fluorescent measurement, a light source 77and beam splitter 78 combine to focus light from the light source 77through suitable optics (not shown) through the wall 76a onto particles(such as indicated by dashed line 79) in the aperture or sensing zone74. Assuming that the particle has been suitably prepared by staining orthe like, fluorescent light emitted by the particle is focused throughthe suitable optics (not shown) on an optical detector 81. Thus far, theoptical system functions like that shown and described in reference toFIG. 13. Additionally, however, provision can be made for simultaneouslymaking light scatter measurements with respect to the particle or sample79. In this, provision can be made for a light source 82 and beamsplitter 83 to couple suitable light, such as red laser light, throughthe wall 76a of the aperture 74. Multiple detectors, such as illustratedby optical detectors 84, 86 and 87 can be positioned at various anglesθ1, θ2, and θ3 around the periphery of the elements defining thetransducer for respectively detecting, for example, forward lightscatter, 90° light scatter and back scatter. Advantageously, for theforward light scatter measurement, the apex of the triangular aperture(reference numeral 75) functions as a field stop.

By incorporating an arrangement such as schematically shown in FIG. 15into the apparatus of FIG. 6, it can be seen that electronic particlevolume, fluorescence, and light scatter measurements can all be madesimultaneously on the same particle or cell sample in the sensing zoneor aperture.

The important advantages of a triangular orifice or sensing zone inaccordance with the principles of this invention are applicable to notonly combined electronic and optical cytometers as discussed above, butare also applicable to purely electronic flow cytometers. FIGS. 16-19pertain to an embodiment of the invention suitable for use in existingelectronic flow cytometers. In this embodiment a supply reservoirhousing 88 has a supply of electrolyte 89 disposed therein. An inlethousing 91 is provided mounted to the bottom of the supply reservoirhousing 88 and suitably fluid-tight sealed with respect thereto by meanssuch as O-ring 92. The inlet housing 91 has a centrally located inletreservoir 93 which communicates via passageways 94 with the electrolyte89. A sample injector 96 is provided centrally located in the inletreservoir 93. Three truncated pyramids 97, 98 and 99 are adhesivelysecured to each other such that their truncated portions define anaperture or sensing zone 101 which is triangular in shape. As is thecase in the other embodiments of the invention, the angular walls of thetruncated pyramids 97, 98, and 99 define an appropriately configuredinlet zone and outlet zone with respect to the aperture 101. An upperpart of the inlet housing 91 is provided with angular surfaces 91a, 91b,etc., which match the angles on the mating surfaces of the truncatedpyramids 97, 98 and 99, with these truncated pyramids being adhesivelysecured to the surfaces 91a, 91b, etc. An outlet housing 102 is providedhaving a centrally located outlet reservoir 103. The bottom portion ofthe outlet housing 102 is provided with surfaces 102a, 102b, etc., whichmatch the mating services on the truncated pyramids 97, 98 and 99, withthe mating surfaces being adhesively secured. As can be seen in thedrawings, the sample injector 96 is positioned such that samples areinjected into the center of the inlet chamber defined by walls of thetruncated pyramids 97, 98, and 99, so as to traverse through the centerof the aperture or sensing zone 101.

In operation, in a manner known to those skilled in the flow cytometryart, suitable means such as a vacuum source are coupled to the outlethousing 102 for establishing a flow of the electrolyte 89 from thesupply reservoir 88 through the inlet reservoir 93, the aperture orsensing zone 101, and out the outlet reservoir 103. Suitable means suchas electrodes and a source of electric potential are provided forestablishing a current flow through this electrolyte. Samples injectedfrom the sample injector 96 enter the flow sheath of the electrolyte andpass through the central portion of the aperture or sensing zone 101.This changes the electrical current through the aperture 101 and causespulses which are detected by suitable electronics well known to thoseskilled in the flow cytometry art.

The advantages of the triangular aperture are very much applicable tothe electronic measurement in the device shown in FIGS. 16-19. Thetriangulated flow sheath of electrolyte is exceptionally stable in itslaminar flow characteristics through the aperture 101. The sample streamtherefore passes through the aperture 101 in a very laminar manner,without any turbulence. Configuring the angles of the truncated pyramidsdefining the orifice 101 in a manner consistent with the principles ofthis invention produces a smooth transition from a region of low currentflux in the inlet chamber to a region of high current flux in theaperture, and back to a region of low current flux in the outletchamber. This smooth transition decreases the edge effects. Furthermore,in a manner discussed previously, configuring the inlet and outletchambers to have a predetermined cross-sectional area relative to thecross-sectional area of the aperture in accordance with the principlesof this invention, reduces the "effective" electronic length of thesensing zone and yields sharp, well-defined pulses. This produces muchsharper electronic pulses occasioned by the passage of a particle or asample through the aperture or sensing zone.

Referring now to FIGS. 21 through 33, there is presented a number ofexamples of various tissues prepared for DNA and/or electronic cellvolume analysis utilizing one embodiment of a flow cytometer transducerin accordance with this invention. Specifically, this data was obtainedusing the flow cytometer illustrated in FIG. 6.

FIG. 21 is a combined electronic particle volume and fluorescence (530nm) histogram of Duke Scientific 10 micron diameter fluorescent spheres.FIGS. 22-26 show DNA histograms (i.e. fluorescence measurements) ofmouse, rat and human origin. Thus, FIG. 22 is a flow cytometric DNAhistogram of nuclei from Wistar rat liver; FIGS. 23 and 24 are similarDNA histograms of nuclei from normal CF1 mouse kidney and liver,respectively; FIG. 25 is a flow cytometric DNA histogram of nuclei fromnormal human breast tissue; and FIG. 26 is a flow cytometric DNAhistogram of nuclei from a patient with breast cancer.

All of the tissue samples used for the data of FIGS. 22-26 were preparedutilizing a nuclear isolation medium which contained DAPI, a DNAspecific dye. The nuclear isolation medium utilized is disclosed in apatent application filed Apr. 24, 1981 in the name of Jerry T.Thornthwaite, and entitled "Nuclear Isolation Medium and Procedure forSeparating Cell Nuclei". As can be seen from FIGS. 22-26, thecoefficient of variation ranged between 1-2% for the DNA histograms,illustrating the efficacy of the optical aspect of the transducer ofthis invention.

FIG. 27 is an electronic cell volume histogram of Wistar rat livercells, which were enzymatically dissociated with collagenase andtrypsin, and measured with the transducer of this invention.

Examples of combined electronic nuclear volume (as differentiated fromcell volume) and DNA fluorescence are illustrated in FIGS. 28, 29 and31. Again, these samples were prepared using the same nuclear isolationmedium and dye as the samples for FIGS. 22-26.

In FIGS. 30(a) and 30(b), 256 channel single parameter data of CF1 mouseliver nuclei by electronic nuclear volume and DNA fluorescence is shown.These data illustrate the application of the transducer of the presentinvention in the simultaneous analysis of a variety of samples utilizingfluorescent and electronic impedance measurements.

FIGS. 31 and 32 further illustrate the simultaneous nature of theelectronic nuclear volume and DNA fluorescence measurements possiblewith the transducer of this invention. FIGS. 31 and 32 are reproductionsof dual channel oscilloscope tracings of electronic and opticalmeasurements on mouse liver. Again, the mouse liver tissue samples wereprepared using the same nuclear isolation medium and dye as the previousexamples, and the optical measurements were of fluorescence. As can beseen from these Figures, the transducer of the present invention trulyresults in simultaneous electronic volume and optical measurements.

Although various aspects of the present invention has been described andillustrated with reference to exemplary embodiments, it is not meant tolimit the invention to these specific embodiments. Various modificationsare possible, and within the skill of those working in this art, withoutdeparting from the true spirit and scope of the invention.

What is claimed is:
 1. A flow transducer comprising means defining anaperture having an axis, said aperture having at least one flat side,means defining an inlet chamber and an outlet chamber immediatelyadjacent the aperture along its axis, at least one of said inlet andoutlet chamber having walls disposed at an angle of at least 5° relativeto the plane of the aperture, said at least one of said inlet and outletchambers at a distance from the aperture of twice the width of theaperture in a plane through its axis having a cross-section area atleast 10 times the cross-sectional area of said aperture.
 2. Atransducer for a flow cytometer in accordance with claim 1 wherein saidtransducer is formed of a plurality of solid polygons joined such thatadjacent flat surfaces of said polygons define walls of the aperture forthe flow cytometer, and others of the surfaces of said polygons definesaid at least one of said inlet and outlet chambers.
 3. A flow cytometerfor making simultaneous electronic and optical measurements on aparticle flowing through a sensing zone thereof, comprising a transducerformed of a plurality of solid polygons joined such that adjacent flatsurfaces of said polygons define a polygonal aperture at the sensingzone and other surfaces of said polygons define inlet and outletchambers to said aperture, at least one of said inlet and outletchambers having a predefined geometric relation to said aperture, saidinlet and outlet chambers defining an arch shaped fluid passageway withthe aperture at the arch vertex, means for establishing a flow ofelectrolyte through said aperture, said inlet and outlet chambers beingconfigured to establish laminar flow of said electrolyte through saidaperture, injector means for injecting samples of particles into saidlaminar flow of electrolyte, one or more electrodes coupled to saidinlet chamber and one or more electrodes coupled to said outlet chamber,means for establishing a current flow through said aperture between saidelectrodes, monitoring means for monitoring the electrical current flowthrough said aperture, at least one of the solid polygons defining saidaperture being an element in an optical measurement system, said systemincluding means for introducing exciting light through said at least onesolid polygon into said aperture, and means for collecting light from asample particle in said aperture.
 4. A flow cytometer in accordance withclaim 3 wherein the predetermined geometric relation between said atleast one of the inlet and outlet chambers and the aperture is such thatwalls of said at least one of the inlet and outlet chambers join theaperture at an angle equal to at least 5° with respect to the plane ofthe aperture, and in which at a distance in the said at least one ofinlet and outlet chambers from the aperture equal to twice the width ofthe aperture along any plane through its axis, the cross-sectional areaof said at least one of the inlet and outlet chambers being at least tentimes the cross-sectional area of the aperture.
 5. A flow cytometer formaking simultaneous electronic and optical measurements on a particleflowing through a sensing zone thereof, comprising a transducer formedof a plurality of solid polygons joined such that adjacent surfaces ofsaid polygons define an aperture at the sensing zone and other surfacesof said polygons define inlet and outlet chambers to said aperture atleast one of said inlet and outlet chambers having a predefinedgeometric relation to said aperture, said inlet and outlet chambersdefining an arch shaped fluid passageway with the aperture at the archvertex, means for establishing a flow of electrolyte through saidaperture, said inlet and outlet chambers being configured to establishlaminar flow of said electrolyte through said aperture, injector meansfor injecting samples of particles into said laminar flow ofelectrolyte, one or more electrodes coupled to said inlet chamber andone or more electrodes coupled to said outlet chamber, means forestablishing a current flow through said aperture between saidelectrodes, monitoring means for monitoring the electrical current flowthrough said aperture, at least one of the solid polygons defining saidaperture being an element in an optical measurement system, said systemincluding means for introducing exciting light through at least onesolid polygon into said aperture, and means for collecting light from asample particle in said aperture.
 6. A flow transducer comprising meansdefining an aperture having an axis, means defining an inlet chamber andan outlet chamber immediately adjacent the aperture along its axis atleast one of said inlet and outlet chambers having walls disposed at anangle of at least 5° relative to the plane of the aperture, said atleast one of said inlet and outlet chambers at a distance from theaperture of twice the width of the aperture in a plane through its axishaving a cross-sectional area which is rotationally asymmetric withrespect to the axis of the aperture and which is at least 10 times thecross-sectional area of said aperture.